This invention relates to a plasminogen activator inhibitor and, more particularly, to cDNA clones representing essentially a full size plasminogen activator inhibitor of the placental type and to expression of the recombinant protein in prokaryotic and eukaryotic hosts.
The plasminogen activators are a class of serine proteases that convert plasminogen to the fibrinolytically active enzyme plasmin (fibrinolysin). Upon being thus activated, the plasmin can attack the coagulation proteins of the fibrin clot (thrombus) and thereby disintegrate the clot. Inhibitors normally present in the blood with plasminogen generally retard this reaction.
Human plasma contains two plasminogen activators that are immunologically distinct, namely tissue plasminogen activator (t-PA) and urokinase (u-PA). t-PA has been demonstrated to have higher affinity for fibrin than u-PA and, therefore, is a preferable agent for degradation of the fibrin clot. The source of the plasma t-PA has been presumed to be the vascular endothelium.
Plasminogen activator inhibitors (PAI) have been obtained from various sources. They are now classified in at least three immunologically different groups: protease nexin-I, the endothelial cell type plasminogen activator inhibitor (PAI-1), and the placental type plasminogen activator inhibitor (PAI-2).
Protease nexin-I was isolated from human fibroblasts and has an apparent M.sub.r 43 kilodaltons (kDa). Scott et al., J. Biol. Chem. 260 (11), 7029-7034 (1985). It is distinguished by its acid lability, its ability to inhibit both plasminogen activators and plasmin, its relatively high pI (7.5-7.8) and by the stimulatory effect of heparin on its activity.
Endothelial cell-type PAI-1 is a glycoprotein of M.sub.r 50-54 kDa that rapidly inactivates both t-PA and .mu.-PA. It is synthesized by endothelial cells and certain hepatoma and fibrosarcoma lines. It is found in platelets and is believed to constitute the major PAI of normal human plasma. See Pannekoek et al., The EMBO Journal 5 (10), 2539-2544 (1986); Andreason et al., FEBS Lett. 209 (2), 213-218 (1986); Ginsburg et al., J. Clin. Invest. 98, 1673-1680 (1986); and Wun and Kretzmer, FEBS Lett. 210, 11-16 (1987).
Placenta-type PAI-2 is a distinct protein of M.sub.r 47,000. Kawano et al., Nature 217, 253-254 (1968); Astedt et al., Thromb. Haemostasis 53, 122-125 (1985); and Wun and Reich, J. Biol. Chem. 262, 3646-3653 (1987). It appears to be immunologically and biochemically identical to a similar activity of human monocytes Chapman et al., Cell 28, 653-662 (1982), and Kopitar et al., Thromb. Haemostasis 54, 750-755 (1985)], and monocytic cell lines [Vassalli et al., J. Exp. Med. 159, 1653-1668 (1984), and Kruithof et al., J. Biol. Chem. 261, 11207-11213 (1986)].
Further background information on the plasminogen activator inhibitors can be had by reference to the recent review article by Sprengers and Kluft, Blood 69(2), 381-387 (1987).
Recent advances in biochemistry and in recombinant DNA technology have made it possible to synthesize specific proteins, for example, enzymes, under controlled conditions independent of the organism from which they are normally isolated. These biochemical synthetic methods employ enzymes and subcellular components of the protein synthesizing systems of living cells, either in vitro in cell-free systems, or in vivo in microorganisms. In either case, the principal element is provision of a deoxyribonucleic acid (DNA) of specific sequence which contains the information required to specify the desired amino acid sequence. Such a specific DNA sequence is termed a gene. The coding relationships whereby a deoxyribonucleotide sequence is used to specify the amino acid sequence of a protein is well-known and operates according to a fundamental set of principles. See, for example, Watson, Molecular Biology of the Gene, 3d ed., Benjamin-Cummings, Menlo Park, Calif., 1976.
A cloned gene may be used to specify the amino acid sequence of proteins synthesized by in vitro systems. RNA-directed protein synthesizing systems are well-established in the art. Double-stranded DNA can be induced to generate messenger RNA (mRNA) in vitro with subsequent high fidelity translation of the RNA sequence into protein.
It is now possible to isolate specific genes or portions thereof from higher organisms, such as man and animals, and to transfer the genes or fragments to microorganisms such as bacteria (e.g., E. coli) or yeasts (e.g., S. cerevisiae). The transferred gene is replicated and propagated as the transformed microorganism replicates. Consequently, the transformed microorganism is endowed with the capacity to make the desired protein or gene which it encodes, for example, an enzyme, and then passes on this capability to its progeny. See, for example, Cohen and Boyer, U.S. Pat. Nos. 4,237,224 and 4,468,464. Likewise, mammalian cells (e.g., mouse, bovine, and Chinese hamster ovary) can be used for the expression of mammalian protein by conventional recombinant DNA methods. See, for example, Axel et al., Science 209, 1414-1424 (1980) and U.S. Pat. No. 4,399,216.
To illustrate, a bacterial plasmid, for example, pSC101 or pBR322 and derivatives thereof, can be used as a cloning vehicle to introduce a foreign or exogenous gene into the host bacteria. An illustrative host bacteria can be, for example, Escherichia coli K-12.sub.x 1776, which is available from the American Type Culture Collection, Rockville, Md. under accession number ATCC 31244. The plasmid can be cleaved with a restriction endonuclease or other DNA cleaving enzyme, for example EcoR I, to form a linear DNA fragment having an intact replicon and cohesive termini. A second DNA fragment having the desired exogenous or foreign gene and a given phenotypical property and complementary ligatable termini can be obtained from a foreign cell or chemically synthesized. This second DNA fragment is spliced with the first DNA fragment with a DNA ligase or other DNA ligating agent, for example T.sub.4 DNA Ligase, to form a completely closed and recircularized plasmid. The insertion of the second DNA fragment into the EcoR I site of the illustrative plasmid brings the expression of the genetic information under the control elements of the plasmid. The resulting recombinant plasmid is then used for transformation of the bacterial cell and allowed to replicate by growing the bacteria in a suitable culture medium. The desired transformants are then isolated by phenotypical trait differentiation, for example, by resistance to particular growth-inhibiting materials such as antibiotics or by various morphological property differences.